C1000 touch thermocycler cfx96 real time system

RNA extracted from liver tissues of infected mice was used for reverse transcription with iScript Reverse Transcription Supermix (BioRad #1708841) to generate cDNA using a MiniAmp Plus Thermal Cycler (Applied Biosystems) with the following reactions: 25°C for 5 min, 46°C for 20 min, and then 95°C for 1 min. Generated cDNA was used for qPCR with iTaq Universal SYBR Green Supermix (BioRad #1725121) and PrimePCR assays for IFNα1, IFNβ1, IFIT1, or ISG15 (BioRad #10025636). qPCR was run on a C1000 Touch Thermo Cycler/CFX96 Real-Time System (Bio-Rad) with the following conditions: 94°C for 2 min, 94°C for 15 s, and then 40 cycles of 55°C for 15 s and 68°C for 15 s.

Total RNA was extracted using an RNeasy Plus Micro Kit (QIAGEN, 74034) according to the manufacturer’s instructions^{15 (link)}. Cell lysates were spun using the kit’s gDNA Eliminator spin columns to remove genomic DNA. Total RNA was purified using RNeasy MinElute spin columns. First-strand cDNA was then synthesized from 1 µg of RNA using PrimeScript RT Master Mix (Takara, RR036A). Then 20 µl reactions were prepared by combining 4 µl of PrimeScript RT Master reaction mix, 2 µl of gene-specific enhancer solution, 1 µl of reverse transcriptase, 1 µl of gene-specific assay pool (20 ×, 2 µM), and 12 µl of RNA diluted in RNase-, DNase-, and genomic DNA-free water. We performed qPCR using TB Green Premix Ex Taq II (Takara, RR820Q) on the C1000 Touch Thermocycler CFX96 Real-Time System (Bio-Rad). Analysis was performed using Bio-Rad CFX Manager software 2.0. The data were normalized to RNA GAPDH and the fold change was calculated via the 2^−DDCt method. The relative concentrations of mRNA were expressed in arbitrary units based on the untreated group, which was assigned a value of 1.

Total RNA was extracted using a QIAGENan RNeasy Plus Micro Kit (QIAGEN, 74034) according to the manufacturer’s instructions. Cell lysates were spun through using the kit’s gDNA Eliminator spin columns to remove genomic DNA. Total RNA was purified using RNeasy MinElute spin columns. First-strand cDNA was then synthesized from 1 µg of RNA using the PrimeScript™ RT Master Mix (Takara, RR036A). Then 20 µl reactions were prepared by combining 4 µl of PrimeScript™ RT Master reaction mix, 2 µl of gene-specific enhancer solution, 1 µl of reverse transcriptase, 1 µl of gene-specific assay pool (20×, 2 µM), and 12 µl of RNA diluted in RNase-, DNase-, and genomic DNA-free water. Quantitative real-time PCR was performed qPCR using TB Green Premix Ex Taq II (Takara, RR820Q) on the C1000 Touch Thermocycler CFX96 Real-Time System (Bio-Rad). Analysis was performed using Bio-Rad CFX Manager software 2.0 (Bio-Rad). The data were normalized to RNA GAPDH and the fold change was calculated via the 2-DDCt method. The relative concentrations of mRNA were expressed in arbitrary units based on the untreated group, which was assigned a value of 1. The primers, which were synthesized and desalted, were from Beijing Genomics Institution, and are shown in Supplementary Data 4.

Total RNA was extracted from FACS isolated cells using TRIzol Reagent (Invitrogen) according to manufacturer’s instructions. RNA was quantified using NanoDrop spectrophotometer and reverse transcribed using High Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific). Gene expression was measured by quantitative RT-PCR using SYBR Green Master Mix (Thermo Fisher Scientific) and a C1000 Touch thermocycler-CFX96 Real Time System (Biorad). Primers spanning exon-exon junctions were used (col1a1 Fw: TCC GGC TCC TGC TCC TCT TA; col1a1 Rev: GTA TGC AGC TGA CTT CAG GGA TGT). The level of each transcript was measured using mouse HPRT (hypoxanthine-guanine phosphoribosyltransferase) mRNA levels as normalizer (mHPRT Fw: AAC TGG AAA GAA TGT CTT GAT TGT; mHPRT Rev: GAA TTT CAA ATC CAA CAA AGT CTG G).

Total RNA was extracted and purified from cultured cells or tissues using the RNeasy Plus Mini Kit (QIAGEN, 74136) according to the manufacturer’s instructions. The RNA was quantified by determining absorbance at 260 nm. One microgram of total RNA from each sample was reverse-transcribed into cDNA using the iScript cDNA synthesis kit (Bio-Rad, 170-8891) in a volume of 20 μl; cDNA from cell samples was amplified. The qPCR was performed using SsoFast EvaGreen Supermix (Bio-Rad, 172-5204) on the C1000 Touch Thermocycler CFX96 Real-Time System (Bio-Rad) according to the manufacturer’s protocol. Analysis was performed using Bio-Rad CFX Manager software 3.1 (Bio-Rad). The specific primers were listed in Supplementary Table 1. The gene expression was calculated via the 2^−ΔΔCt method and normalized to 18SRNA/18srna^{72 (link)}. The relative concentrations of mRNA were expressed in arbitrary units based on the untreated group, which was assigned a value of 1.

We designed specific primers for the two Tn5 mutants in this study, which included one primer annealing to the Tn5 insertion [28 (link)], with the second primer designed to anneal to the flanking genome region (Table S1). For the wild-type phenotype M. japonicum, we utilized a strain containing DsRed integrated into the genome (M. Hayashi’s personal communication; [29 (link)]), which allowed us to use DsRed primers to identify the wild type [30 (link)]. These primers were used throughout to estimate the proportions of each genotype in vitro and in planta via qPCR (Table S1).
qPCR was performed in a total volume of 25 µL. Each reaction contained 1 µL of template DNA, 1× SYBR Green qPCR master mix (Bio-Rad; Hercules, CA, USA), and primers at a final concentration of 0.4 µM. Reactions were run on a BioRad CFX96 Real-Time System C1000 Touch Thermocycler with the following settings: initial warm-up at 95 °C for 3 min followed by 40 cycles of denaturation at 95 °C for 15 s, annealing at 58 °C for 20 s, and extension at 72 °C for 30 s. A melt curve was generated from 65 °C to 95 °C with an increase of 0.5 °C every second.